Ingesting infective Sarcocystis neurona sporocysts has been determined to be the cause of equine protozoal myeloencephalitis (EPM). S. neurona has a host life cycle stage consisting of natural prey species, an intermediate host and a definitive host. The opossum has been determined to be the definitive host species. The feral opossum (Didelphis virginiana) and the South American opossum (D. albiventris) consume the intermediate host's muscle tissue infected with protozoal sarcocysts. Following ingestion, the protozoa undergo sexual reproduction in the intestinal epithelium of the host opossum to form oocysts. Stimulated by the intestinal environment, the oocysts undergo sporulation producing sporocysts that are eventually shed through the host opossum's feces. The speculated transmission route between opossum and equines is by fecal-oral transfer through contaminated feed or water ingested by horses.
Equines are an aberrant host because the ingested sporocysts mature into the merozoite life cycle stage but do not form sarcocysts in horse's muscle tissue. Following excystation, the sporozoites penetrate the intestinal mucosa of the horse, and undergo a series of replicative cycles in the vascular endothelial cells, and possibly in the white blood cells. The merozoites then migrate to the central nervous system where they continually divide without encysting (i.e., they do not form cysts). The merozoites divide by polygeny and often leave a residual body that gradually destroys the nervous tissue of the infected horse causing spasticity, hypermetria, ataxia, paralysis, recumbency, and death. The life-cycle stage of the protozoa that is found in horses cannot be transmitted to other horses nor can the tissue of horses, even if eaten by opossums, infect the opossum. Therefore, the horse is a dead end host for the protozoa.
The current methods of detection of EPM involve the analysis of cerebrospinal fluid for the presence of anti-S. neurona antibodies, as well as cytologic analysis. Antibodies are detected by an immunoblot test developed by Granstrom, D. E. (Diagnosis of equine protozoal myeloencephalitis: Western blot analysis. 1993. Proc. Eleventh ACVIM Forum: 587-90). Antibodies can also be detected by FIAX, a modified immunofluorescent antibody cross-reaction test. This test is performed using Sarcocystis cruzi bradyzoite antigen, and relies on cross-reacting antibodies. A PCR/DNA test has also developed for detecting possible protozoal infections in the central nervous system of horses, but this test is generally not useful in the diagnosis of EPM. (Fenger C K, Granstrom D E, Langemeier J L, Stamper S. Detection of Sarcocystis neurona in cerebrospinal fluid of horses by nested polymerase chain reaction. J. Vet. Diagn. Invest., in press, c).
Current treatments for EPM involve the use of antibiotics that inhibit replication of the protozoa. However, there presently are no FDA or USDA approved drugs for this disease. As a consequence, there have been a number of products made available by either personal foreign importation or from off-label sources. The use of antibiotics presents two problems, a short-term course of antibiotics may only send the protozoa into remission and a long-term exposure can allow the protozoa to adapt and become resistant to the treatment. As a result, EPM prevention and treatment has become a major focus in the equine industry.
The currently available treatments for EPM are expensive, of limited efficacy, and may include adverse side effects such as anemia, abortion, diarrhea, low white blood cell counts or the like. As with most diseases, veterinary practitioners indicate that prevention of this disease is the optimal solution.
Vaccines derived from merozoites of Sarcocystis neurona for prevention of EPM have recently become available to protect equines against infection. To determine the efficacy of the vaccine strains, studies are conducted to determine resistivity in challenged animal models. The animals are orally exposed to the protozoa and the resultant infection is quantified by observation of clinical symptoms and examination of central nervous system tissue samples.
To quantify the viability of sporocysts, samples are excystated in vitro. Current chemical excystation methods are time consuming and involve expensive reagents that mock the harsh conditions of the intestine that are believed to stimulate sporocyst development. These procedures involve multiple washing steps because of the harsh chemicals and lengthy incubation periods, for instance, see Murphy, A. J. et al. “Simplified Technique for Isolation, Excystation, and Culture of Sarcocystis Species from Opossums,” J. Parasitol., 1999, p. 979-981, Vol. 85(5), American Society of Parasitologists. R. J. Cawthorn et al. teaches excystation methods of S. cruzi, S. tenella, and S. capracanis in “In Vitro Excystation of Sarcocystis capracanis, Sarcocystis cruzi and Sarcocystis tenella”, J. Parasitol., 1986, p. 880-884, Vol. 72(6), American Society of Parasitologists, but does not detail a generalized method for S. neurona. J. P. Dubey et al. in Sarcocystis of Animals and Man, 1989, CRC Press, Inc., Boca Raton, Fla., notes other procedures that involve expensive excystation reagents such as trypsin, bile salts, and chelating agents such as sodium ethylenediamine tetracetic acid (EDTA). Using these excystation reagents requires tedious washing of the sample to remove the excess solution in order to preserve the sporocysts.
There thus exists a need in the art for a simpler, less time-consuming, and inexpensive method for excystation of sporocysts. Also needed are new compositions that can serve as excystation fluids.